title: The path of the growing peptide chain through the 23S rRNA in the 50S ribosomal subunit; a comparative cross-linking study with three different peptide families.
abstract: As part of a programme to investigate the path of the nascent peptide through the large ribosomal subunit, peptides of different lengths (up to 30 amino acids), corresponding to the signal peptide sequence and N-terminal region of the Escherichia coli ompA protein, were synthesized in situ on E.coli ribosomes. The peptides each carried a diazirine moiety attached to their N-terminus which, after peptide synthesis, was photoactivated so as to induce cross-links to the 23S rRNA. The results showed that, with increasing length, the peptides became progressively cross-linked to sites in Domains V, II, III and I of the 23S rRNA, in a similar manner to that previously observed with a family of peptides derived from the tetracycline resistance gene. However, the cross-links to Domain III appeared at a shorter peptide length (12 aa) in the case of the ompA sequence, and an additional cross-link in Domain II (localized to nt 780-835) was also observed from this peptide. As with the tetracycline resistance sequence, peptides of all lengths were still able to form cross-links from their N-termini to the peptidyl transferase centre in Domain V. A further set of peptides (30 or 50 aa long), derived from mutants of the bacteriophage T4 gene 60 sequence, did not show the cross-links to Domain III, but their N-termini were nevertheless cross-linked to Domain I and to the sites in Domains II and V. The ability of relatively long peptides to fold back towards the peptidyl transferase centre thus appears to be a general phenomenon.
authors.affiliation: Max-Planck-Institut für Molekulare Genetik, AG-Ribosomen, Ihnestrasse 73, 14195 Berlin, Germany.
authors.forename: K M
authors.initials: KM
authors.lastname: Choi
authors.forename: R
authors.initials: R
authors.lastname: Brimacombe
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D003432:Cross-Linking Reagents; D010455:Peptides; D021382:Protein Sorting Signals; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012338:RNA, Ribosomal, 23S